Regulation of tyrosinase mRNA levels in mouse melanoma cell clones by melanocyte-stimulating hormone and cyclic AMP
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Mouse melanoma cells in culture respond to melanocyte-stimulating hormone (MSH) by demonstrating increased activity of tyrosinase, the rate-limiting enzyme for melanin synthesis. Because this stimulation is strictly dependent upon continued transcription and translation, we have carried out studies to determine if MSH increases the level of tyrosinase mRNA. The abundance of tyrosinase message levels in melanoma cells treated with either MSH or dibutyryl cAMP was determined by Northern blot analysis utilizing a 946 base pair mouse tyrosinase cDNA probe. The tyrosinase cDNA was isolated from a λgt11 expression library generated from mRNA isolated from theopylline-induced Cloudman melanoma cells. The abundance of tyrosinase mRNA was determined in an amelanotic cell clone (AM-7AS) and a melanotic cell clone (MEL-11AS). The melanotic cell line had five times as much tyrosinase activity and almost 10 times more tyrosinase mRNA than the amelanotic line. Tyrosinase activity and mRNA increased in both cell lines after MSH addition. The amelanotic line treated with MSH for three days showed a fivefold increase in tyrosinase activity and a twofold increase in tyrosinase mRNA. The melanotic cell line treated with MSH for three days showed a 3.7-fold increase in enzyme activity and an eightfold increase in the abundance of tyrosinase mRNA. Dibutyryl cAMP also stimulated tyrosinase activity and the accumulation of tyrosinase mRNA. The data suggest that MSH, acting through cAMP, promotes an accumulation of tyrosinase mRNA.
KeywordsMelanoma Cell Cell Clone Northern Blot Analysis Tyrosinase Activity Melanin Synthesis
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